Indian Journal of Biochemi stry & Bi ophysics
Vol. 37, December 2000, pp. 44 1-446
L( l ( (
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.
Overexpression and reconstitution of a eske iron sulfur protein
from the
_.
cyanobacterium nechocystis
803
~
(
•
I
-
•
•
Dirk 1 chneider Karin Jaschkowitz, Andreas Seidler and !Matthias Rogner*
L~:stuhl fur Bioc
.
emi e der Ptlanzen, Fakultat fU r Bi ologie, Ruhr-Uni versitat Bochum, D-447800 Boch um , Ge: : . : V
Accepted 23 JUli e 2000
Chl oropl ast cyt b(! co mplexes as well as mitochondrial and bac teri al cyt bCI complexes co ntain a hi gh potenti al Rieske
iro'rr-s ul fur protein whic h is essent ial for their fun ction. To charac teri se the isolated Rieske protei n from the meso phili c cyaI nobacteriu m SYll ecli ocystis PCC680
cloned the encod ing gene into an expression vector and overexpressed the protein
m E. coli. In cell s overexpressing the protein no typical Ri eske type EPR signal was detected neither in membranes nor in
inclu sion bod ies where the majority of the protein was depos ited. The inclusion bod ies were iso lated from the E. coli cell s
and denatu rated with 8 M urea. With a si ngle ani on exchange chro matographic step a pure pro tein cou ld be obtained whi ch
was used fo r furt her ex periments. The NifS like protein IscS was recently reported to mediate the incorporation of iro nsul fur clusters into fe rredoxin ill vitro.
,used the recombinant IscS pro tein for the incorporation of the cluster into the
fo lded Ri eske apoprotein. Spectroscop ic characte ri sation of the resultant pro tein by CD and EPR spectroscopy showed the
presence of a typical Rieske iro n-su lfur ce, : 9 ~ .
~/ _
we
Introduction
Rieske iron-sul fur proteins are essenti al components of cytochro me bCI and cytochrome bel complexes in pro karyoti c and euk aryo tic cell s. They pl ay
an important ro le in th e electron transport chain
within the cyt bc compl exes and medi ate the electron
transfer between a cyt b protein component (cy t b or
cyt b6) and a c-type cytochrome (cy t c, or cyt f) (for a
recent rev iew see ref. I ) . Ri eske proteins contain a
redoxacti ve [2Fe-2S ] cluster whi ch is coordin ated by
two cystein s and two hi stidine res idues of the apoprotein . Two additi onal cysteine res idues, whi ch are
located close to th e cluster binding res idues, form an
intern al disul fide bri dge and are required fo r th e stability of the protein 2. The iron-sul fur clu ster contain-
ing Ri eske protein is characteri sed by typical EPR
spectra' and an unusual hi gh redox potenti al in th e
range of +100 mY to +400 mY (ref. 4).
The three di mensional stru cture of the water soluble part of th e Ri es ke protein from the cyt bc, as we ll
*To whom correspondence should be addressed.
E-mail: Mallh ias.Roegner@ruhr-un i-bochum.de
Abbreviations: CD, circul ar dichro ism; cyt, cytoc hro me; DTT,
di th iot hrei tol; ~-DM , dodecyl ~-D-ma l toside ; EDTA, eth ylenediamine tet raacetic acid; EPR, electron paramagnetic resonance;
IPTG , i so propy l -~-D- th ioga l ac t opyra n oid; PCR, po lyme rase chain
reaction; PMSF, phenylmet hanesulfonyl tluorid; SDS-PAGE,
sodium dodecyl su lfa te-polyac rylamide gel electrop horesis;
TLCK, Tosyll ysylchl orometh ylketone
as of the cyt bJ complex was reso lved up to 1.5 and
1.8 Angs tro m, res pecti ve l/ ·6 . Although th e sequence
similarity between the Ri es ke protein s from cyt bc,
and cyt bJ compl exes is quite low 6 th eir three di me nsional structure apparentl y is very conserved 7 . Their
water so luble part consist of two domains: A small
redox ac ti ve domain binding the iron-sulfur clu ster
and a larger scaffold domain . As all Ri es ke protein s
contain a hydrophobic sequ ence at the N termi nus,
th e exi stence of a transmembrane anchor was discussed for a long time. Indeed, the 3D structure of the
cyt bc, complex clearl y shows a transmembrane hel ix
8
fo r th e Ri eske prote in , sugges tin g strongly a similar
structure for the Ri es ke protein of the analogous cyt
bJ complex. Up to now several attempts have been
made to overex press and characteri ze Ri eske proteins: The Ries ke genes of some hyperth ermophi lic
crenarchaeons were cloned and heterologously expressed in E. co li 4.9; aft er purifi cati on these overexpressed protein s we re shown to contain th e typica l
Ri es ke iron-s ulfur centre. Also, the Ri es ke protein
fro m the mesophilic cyanobac terium Nosfoe
PCC7906 was overex pressed, yielding inclusion
bodi es whi ch di d, however, not co ntain th e ironsul fur clu ster' o. Instead, th e auth ors reco nstituted the
protein by a chemi ca l procedure and were able to
show the typica l spectroscopic properti es of a Rieske
protein.
442
IND IAN 1. BIOCHEM. BIOPIIYS, VOL. 37, DECEMBER 2000
Here we report the overex press ion and purification
of a Ri es ke prote in from the cyanobacterium Synechocystis PCC6803. The functional reconstitution of
the Fe-S-c1uster for thi s Ri es ke protein was for the
first time ac hieved using a reco mbinant NifS like
protein .
Materials and Methods
DNA cloning, seqllencing and gene expression
E. coli strains were routinely grow n in LB medium
suppl emented by 100 Il g/ml Ampicillin (w hen indi cated). For plasmid propagat ion, strain OH5u and for
overexp ress ion strain BL2 1(OE3) was used. Molecul ar standard techniques were performed l2. The
restriction enzy me Nde I was purchased from New
England Bi olabs and all other DNA mod ify ing enzymes from MBI Fermentas. The ge ne encodin g the
Synechocystis Ri eske protein II was amplified by PCR
using the primers NT1316 (5'-G GGAATTCC
AT ATGACCCAGA TTTCTGGCTCCCC) and CT
13 16 (5'-GAGTCGGATCCTTAAGCCCACCAGG
GATCTT AGTCG).
For clonin g the PCR product into the ex press ion
vecto r pRSET6a (ref. 13), a Ndel site was introd uced
5' of th e gene including th e trans lation start codo n
ATG and a BWI1HI recognition site was added 3 ' of
the stop codon of the ge ne. The PCR ampl ified ge ne
was cleaved with Nde I and BWI1HI and cloned into
th e Nde l/BwlIHI side of th e exp ress ion plasmid. For
overexpress ion a freshly transfo rmed BL21 (OE3) E.
coli colony was grow n for 6 hours in 10 ml of LB
medium co ntaining 100 Il g/ml ampicillin . Thi s cul ture was used to inocu late 500 ml of th e sa me medi um . After 15 hours incubati on at 37°C under co ntinu ous shakin g th e ce ll s were harvested.
Purification of th e overexpressed protein
Cell s overex pressing th e Ri eske protein were harvested by centri fugation and inclu sion bodies we re
iso lated l2 . After disso lving th em in urea-buffer (50
mM Tri s- HCI, pH 8.3, and 8 M urea) they were purifi ed on an ani on exc hange paRaS 50 HQ co lum n
(Persep ri ve Bi osystems) using a Bi oCad 700E system
(PE Biosystems). The protein was app lied onto th e
co lumn equilibrated with urea-b uffer and e luted with
a two-s tep NaCI grad ient (0-0. 18 M, 15 ml and 0.18- 1
M, 8 ml) at a flow rate of 3.5 ml/min . The eluted
prote in was co ncen trated by Centricon-l 0 ultrafiltratio n unit s (Am icon). Cell membranes of SYll ecllOcystis and th e cyt b6 f comp lex were preparedl~. Protein
co ncentrati ons were determined I).
Refolding of the protein and reconstitlltion of th e
FeS-Cluster
Refolding of the Ri eske protein was done 10. In detai l, the overprodu ced and purified prote in was incubated for 30 min in 50 mM Tris-HCI, p H 8.3, I % ~
mercaptoeth anol and 0.05 % ~ -OM (a ll buffers have
bee n flu shed by argo n gas pri or to use). Refo lding of
the protein was ac hieved in two ways:
a) The protei n was diluted 8-fold in 50 mM TrisHCI pH 8.3, 0.05 % ~-OM, whi ch was suppl emented
by I mM PMSF and I mM TLCK to avo id protein
degradati on.
b) The protein so luti on was dia lysed for 15 hr
against thi s buffer.
Inco rporat ion of the FeS-c lu ster was performed
enzy mati call y using th e overprod uced and purified
NifS-like Synechocystis protein IscS (ref. 16). I mM
OTT,
I mM
pyrodoxalphosphat e,
2
rnM
Fe(NH~hCSO~h 10 mM L-cystein and 6 Il g IscS were
added und er stri ctl y anae robic co ndit ions and t he soluti on was incubated for 2 hr at 30°C. One vo lume of
th e reconstituti on mi xture (typ ically 5 ml) was dialysed for 15 hr against 100 vo lumes of 50 111M
TrisHCI, pH 8.3, 0.03 % ~-OM, I 111M PMSF, I rnM
TLCK and 10 mM EDT A. Samp les we re concentrated to a final vo lume of I ml on Ce ntri con -I () ultrafiltration units (A micon).
SOS PAGE and immun ological analysis was routin ely done l2 . For detection of th e overexpressed
protein a peptide antibody directed against the protein
N-terminus was used.
Spectroscopic characterisation of th e reconstitllt ed
protein
Circular dichroism spectra (ro utin ely between 300
nm and 600 nm) we re recorded on a J asco 7 15 CD
spectrometer, using a slit width of 2 nm ; th e signal to
noise rati o was improved by a 10-times acc umulati on
of the spectra.
EPR spec tra we re reco rded at the Sec ti on de Bi oenergetiqu e, CEA-Sac lay, France, on a Bruker EPR200
spectrometer; a helium cryostat (Oxford In struments)
kept the sample temperature at 15K. Other parameters for the measurement include 6.3 mW mi crowave
power, 10 G mod ul ati on amplitude and 9.44 GHz microwave frequency.
Results
Overproduction of th e SynecllOcystis Rieske protein
A plasmid for th e overexpress ion of th e Synec!lOcystis Ri eske protein was ge nerat ed by PCR-
SCHNEIDER el a l.: OVEREXPRESSION AND RECO STITUTION OF RIESK E IRON SULFU R PROTEIN
amplification of the petC gene; the resultin g 540 bp
DNA fra bGment was cloned into the express ion vec tor
pRSET6a and sequenced. Thi s plasmid was used for
overex pressi on of the Syn echocystis Ri es ke protein.
For analysing the success of the overexpress ion,
membranes from SYll echocystis, purified cyt brJ co mpl ex of SYllechocystis and E. coli cell s were separated
by SDS-PAG E, followed by immuno-bl ottin g. Fi g. I
shows th e cross reacti on of the Ri es ke antibody with a
19 kDa band in the th ylakoid membranes of Synechocystis (lane I) and al so with the iso lated cyt brJ co mplex (lane 2). The apparent molecular mass of 19 kDa
agrees we ll with the molecular weight of th e Ri es ke
protein as detcrmined from th e correspondin g amin o
acid sequence. As th e se parated E. coli protein s show
a band of identi cal apparent mass (lane 3), crossreactin g with the same antibody, thi s indica tes the successful overex press ion of th e Ries ke protein. The recombinant protein accumulated to about 15 % of th e
total E. co li cell protein s. Further an alys is showed
that th e protein is located exclusive ly in the inclusion
body frac ti on of E. coli .
Purific([tion of th e ove rp rodll ced protein
Since ne ither the inclu sion bodi es iso lated from E.
coli nor the E. coli membranes showed EPR signals
typica l for iro n-sulfur clusters we tri ed a different
strategy by reco nstitutin g the iron-sulfur clu ster into
the co rrectl y folded apoprotein .
Although th e inclusion bodies are hi ghl y enri ched
in th e ove rex pressed protein furth er purificati on of
the Ri es ke protein was required. The inc lusion bodi es
from E. coli were denatured with 8 M urea and th e
reco mbinant Ri es ke protein was purified by ani on
exchange chromatograph y; Fi g. 2 shows a typical
HPLC eluti on pro fil e: Whil e most E. coli proteins copuri fied with the inclusion bodi es did not bind ont o
the co lumn , th e overex pressed Ri es ke protein eluted
at about 20 mM NaCI , yie lding about 10 mg Ri es keprotein . Fi g. :\ shows overexpressed Ri es ke protein at
di fferent stages of puri fi cat ion, revea led on S DSPAG E.
Refoldin g of th e Rieske protein and in co rpo ration of
th e FeS ellIster
Reco nstitut ion of th e R ieske protein was achi eved
in two steps: First the HPLC purified Ri es ke protein
had to fold co rrec tl y, and then th e iro n-sulfur c luster
coul d be inco rporated. Fo r th e foldin g of the protein
two meth ods ha ve bee n appli ed successfully : In th e
1
2
443
3
.., 'I i
• ••
Fig. I- Overex pression of the full length S\'II ecllOc."slis Rieske
protein in E. coli [Immunoblot of a SOS-gel of SYll echocyslIS
membranes (lane I ), purili ed cyt b(/ co mpl ex (lane 2) and cell
ex tract of E. coli overexpressing the Ri es ke protein (lane 3) . The
specilic anti body directed against the N-termin us of the SYll echo cysl is Rieske protein shows the presence of the protein in all three
samples].
first one the soluti on of the denatured protein was
qui ckly dilut ed 10-fold in a buffer without urea; and
in th e second one, the protein so luti on was di alysed
ove rniGht
aG
b
b ainst a buffer without urea resultin g in a
slow urea removal. Both procedures did not show
siG
nificant difference in the amount of reconstituted
b
.
Ri es ke protein . After refoldin g the apoprotelll was
used for the incorporati on of the iron-sulfur clu ster
simil ar to th e method reported in ref. 16 .
The inserti on of th e iron-s ulfur c luster into the Ri es ke apoprotein was chec ked by spec troscopic met hods. Figure 4 shows th e CD spectrum of the reconstituted Ri es ke protein with two band s at 326 nm and
394 nm. Although max ima and minima differ sli ghtl y
in their positions, the overall spectrum is in good
agreement with the CD spec trum of th e cy t bc I Ril7
es ke protein indicating a successful reconstituti on.
Ori ginally , EPR spec troscopy was used for the characteri zation of the iron-sulfur clu sters l8 , and th e EPR
spec trum of the reco nstituted Ri es ke protein fro m
Synechocystis is shown in Fig. 5. The spectrum shows
the typical g va lues for Ri es ke proteins with gz= 2.04
and gy= 1.90. As th e gz area is broad, the co rrect va lue
is diffi cult to determin e from thi s EPR spec trum .
However, the similarity of thi s spectrum with the
EPR spec trum obtained from iso lated SyneellOcystis
cyt bGf complex 19 and other Ri es ke proteins strongly
sUGGests
that the overprodu ced Ri eske protein was
bb
.
correctly refolded and the iron sulfur c luster was tn corporated.
Discussion
Overexpression and pur(fic([tion of th e recolI/binont
Rieske protein
Although E. coli is th e most popul ar hos t organism
.
.
10 I
for heterologous ex press ion of fo retgn
protellls, Ile
overexpress ion of membrane prote ins in E. coli ofte n
444
INDI AN J. BIOCHEM . BIOPHYS., VOL 37, DECEM BER 2000
1.0~------------------------------------------~
E
c
o
CX)
N
.....CO
Q)
g 0.5
-
CO
..0
0.5 u
..0
Z
oCJ)
co
«
o
0.0
o
5
10
15
20
Time / min
Fig. 2-Pu ri ticat ion of overex pressed and enriched Ri eske pro tein by an ion-exchange chromatograp hy. IE lu tion profil e of a typi cal anion-exchange I-IPLC run (g ray line, peak of Ri eske protein shaded) with a NaCI gradient from 0-1 M (black line). Absorbance was recorded at 280 nm] .
leads to cell death caused by the toxicity of the overex pressed protein . While this toxi c effect is often
caused by th e association or incorporati on of the recombinant protein into E. coli membranes, th e toxicity often decreases when the protein is located in inclusion bodi es. As th e formati on of inclu sion bodies
results primaril y from hydroph obi c interactions, the
success ful overex press ion of a protein with only one
potential transmembrane helix like the Ri eske protein
seems reali sti c. There are examples for the integrati on
of overexpressed and correctly fold ed membrane
proteins into E. coli cel l membranes 2o, and also the
overexpression of a correctly fold ed Rieske protein
with an incorporated iron-sulfur cluster has been reported49·. However, the overexpressed, full length
Ri eske protein of Synechocystis was only found in the
inclu sion body fract ion which did not show any typical EPR Ri eske signal after so lubilizat ion in nondenaturating buffer; thi s indicates that th e protein is
overex pressed in an denatured form. Since the references given above show the poss ibility to incorporate
an iron-s ulfur cluster into Ri eske proteins overexpressed in E. coli , the misfolding of the apoprotein
should be th e reason for th e absence of a Rieske EPR
signal. It should be pointed out, however, that th ese
references on the one hand refer to the overexpress ion
of Ries ke proteins from thermophilic crenarcheo ns4.9;
on the other hand, Holton et al. 10 mention th e presence of overexpressed Ri eske protein from the cyanobacterium Nostoc PCC7906 in E. coli membranes,
but they were not able to detect any Ri eske Ironsulfur cluster in the membrane fraction.
The inclusion bod ies puri fied form E. coli cell s
overexpressing the Syn echocystis Ri eske protein were
hi ghly enriched with the reco mbinant protein , and the
denaturated protein was obtained in an essent iall y
pure form by only one additional pu rificati on step
which allowed further reconstituti on experiments
(Fig. 2). It should be be noted th at we were un ab le to
obtain reconstituted Ri eske proteins fro m so lu i1i sed
inclusion bodies unl ess the Ri eske prote in was furth er
solubilized. Thi s indicates that other proteins colocated in the inclu sion bodies may interact with th e
Ri es ke protein , thereby interfering e ither with the
folding of the protein or with the incorporation of the
iron-sulfur cluster.
SCH NE ID ER el
MW
a /.:
OVEREXPRESSION AND RECONSTITUTION OF RI ES KE IRO N SULFUR PROTEIN
CE
IB
445
2,....-------------,
IEC
40 kDa
....
r:I\
~
30 kDa
"CI
E 0
Q
U
20 kDa
•
-1
10 kDa
-2+----+----+---~
Reconstitutioll of th e overexpressed protein
In principle, the recon stitution procedure for the
Rieske protein developed in thi s paper is derived
from the one reported for the cyanobacterium Nostoc
PCC7906 (ref. 10), whi ch was the first one for any
Rieske protein : In the first step the protein is allowed
to fold correc tl y and then the iron-sulfur cluster is
incorporated. However, different from Holten et al.
who used a chemical procedure for the incorporation
of the cluster, we applied an enzymatic approach for
the iron-sulfur cluster formation. The NifS-like protei n IscS from Synechocystis was recentl y show n to
be ab le to mediate the inco rporati on of iron-sulfur
clusters into ferredoxin ' 6 in vitro by ac ti vati on of su lfur from cysteine. Instead of ferredoxin , we used thi s
meth od for th e reconstituti on of the refold ed Ri es ke
proteins and probed the successful reconstitution by
the characte ri stic CD and EPR signals. Further experiments which are in progress should show whether
this IscS protein plays a role in vivo also in the maturation of the Ri es ke protein.
500
400
300
Fig. 3-S DS-PAGE of the overexpressed Rieske protein at dilTerent stages of purillcati on. [MW, molecul ar mass standard; CE,
total E. coli protein ; IB , inclu sion bodies; IEC, Rieske protein
after anion-exchange chromatographi c purillcation] .
600
Wavelength [nm]
Fig. 4-Vi sible CD spectrum of the reconstituted Rieske protein
in reduced state. [The spectrum was recorded at 25 °C in 50 mM
Tris-HCI p H 8.3. System settings : Bandwidth, 2 nm; scan speed,
50 nm/min; res ponse, 4 sec; reso lu tion , 2 nm ; acc umul ation , 10J.
9z=2.04
I
3100
3300
9y =1 .9 1
I
3700
3500
Magnetic field [G]
3900
4100
Fig. 5-EPR spectra of the reconstituted Rieske protein in reduced state. For experimental co nditions see Materials and
Methods.
INDIAN J. BIOCHEM. BIOPHYS ., VOL. 37, DECEM BER 2000
446
The re are several reports on th e chemical reco nstitution o f all-cysteine ligated iron-sulfur c lu ste rs
into apoproteins in th e lite rature" I - 23 . Also, for some
prote ins with cysteine ligated iron-sulfur c lusters, it is
shown that IscS can be used for recon stitution 16. Our
re port shows for th e first time an involveme nt of IscS
in the formation of a cys teine/hi stidine ligated ironsu I fur cluster ill vitro.
Th e gz and gy values of the clu ste r, gz= 2 .04 and
gy= 1.90 , as dete rmined from th e EPR spectra , are in
good agree me nt with the values obtained from th e
whole co mplex 19 . These data indi cate that the g values of th e Ri es ke centre are inde pe nde nt of th e procedure used to in corporate the protein into the c yt brJ
co mpl ex.
It is known that Ri es ke prote in s are involved in
fo rmation of th e plastoquin o le oxidation site of th e
cyt be co mpl exes and also in inte raction s of th e ir lumin a l part with other su bunits of the complex2~. Althou gh th e iron-sulfur cluster is located in the e xtrinsic domain on th e Ri eske prote in , inte rac tion s betwee n th e luminal prote in part and other proteins with
poss ibl e e ffec ts o n the g values cannot be excluded .
As th e iso lated, reconstitut ed Rieske protein from
Sy ll ec/weysfi s shows the same EPR s ignal as the cyt
bel complex with a n integrated Ries ke protein , o ur
results indicate th at the spec troscop ic prope rti es of
thi s subunit are indepe ndent of its in co rporation into
th e cyt bol complex.
Th e successful ill vitro reco nstituti on of the SYll eehoeysfis Ri es ke prote in prese nted in thi s pape r is an
excell e nt basis for furth e r characterisation o f th e s pec ifi c rol e of thi s subunit, es pecially for th e study of
structure/func ti o n re lation s invol vin g s ite direc ted
mutati ons.
2
3
Davidson E, Ohni shi T, Alla-Asafo-Adjci & Daldal F ( 1992)
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17
Komp lexes aus gcrichtcten Mut anten dcs Cyanobaktcriums
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We would like to acknowledge A .W . Ruthe rford
and A . Bou ssac for giv ing the poss ibility to use the
EPR s pectromete r at CEA Sacley. This work was
supported by th e Deutsche Forschungsgeme in sc haft
(Gradui e rte nkoll eg "Biogenese und M ec hanisme n
kompl exer Zellfunktionen"), the Human Frontier Science Program (OS and MR) and the Ministry of Scie nce & Resea rch of Northrhin e Westphalia (A S) .
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